High‐Performance Liquid Chromatographic Determination of Parecoxib in Human Plasma and Pharmaceutical Formulations

Abstract

A simple and sensitive RP‐HPLC method for the determination of parecoxib (PXB) in human plasma and pharmaceutical formulations has been developed and validated. The separation of PXB and the internal standard, ibuprofen (IBF) was achieved on a CLC C₁₈ (5 μ, 25 cm×4.6 mm i.d.) column using UV detector at 200 nm. The mobile phase consisted of acetonitrile‐water (92:8 v/v). The linear range of detection was found to be 0.9–18.4 µg/ml (r=0.9985). Intra‐ and inter‐day assay relative standard deviations were observed to be less than 0.3%. The method has been applied successfully for the determination of PXB in spiked human plasma and pharmaceutical preparations. Analytical parameters were calculated and complete statistical evaluation is incorporated.     

Post‐chemiluminescence Method of the Determination of Loperamide Hydrochloride in Human Plasma and Pharmaceutical by Using Dichlorofluorescein as Chemiluminescence Reagent

Abstract

A post‐chemiluminescence (PCL) was observed when loperamide hydrochloride solution was injected into the reaction mixture after the finish of CL reaction of alkaline N‐Chlorosuccinimide (NCS) and dichlorofluorescein. Based on this phenomenon, a simple, sensitive and fast flow injection PCL method was established for the determination of loperamide hydrochloride. The possible mechanism for the PCL reaction was discussed via the investigation of the CL kinetic characteristics, the CL spectra, the fluorescence spectra. The PCL intensity responded linearly to the concentration of loperamide hydrochloride in the range 8.0×10^?10 to 6.0×10^?7 g · ml^?1 with a linear correlation of 0.9995. The detection limit was 4×10^?10 g · ml^?1. The relative standard deviation was 2.4% for 4.0×10^?8 g · ml^?1 loperamide hydrochloride (n=11). This method has been applied to the determination of loperamide hydrochloride in human plasma and pharmaceutical samples with satisfactory results.     

Liquid Chromatography‐Electrospray Ionization Mass Spectrometric Method for Determination of Gliclazide in Human Plasma

Abstract

A rapid, sensitive, and specific liquid chromatography‐electrospray ionization mass spectrometric (LC‐ESI‐MS) method has been developed for quantification of gliclazide in human plasma. The analyte and tolbutamide (internal standard, I.S.) were extracted from plasma samples with n‐hexane–dichloromethane (1:1, v/v) and analyzed on a C₁₈ column. The chromatographic separation was achieved within 4.0 min by using methanol–0.5% formic acid (80:20, v/v) as mobile phase and the flow rate was 1.0 mL/min. Ion signals m/z 324.0 and 271.0 for gliclazide and internal standard were measured in the positive mode, respectively. The method was linear within the range of 2.5–2000 ng/mL. The lower limit of quantification (LLOQ) was 2.5 ng/mL. The intra‐ and inter‐day precisions were lower than 2.8% in terms of relative standard deviation (RSD). The inter‐day relative error (RE) as determined from quality control samples (QCs) ranged from ?1.93% to 1.85%. This validated method was successfully applied for the evaluation of pharmacokinetic profiles of gliclazide modified‐release tablets in 20 healthy volunteers.     

Rapid and Sensitive LC−MS–MS Method for the Determination of Sarpogrelate in Human Plasma

A rapid and sensitive liquid chromatographic–tandem mass spectrometric method has been developed and validated for the estimation of sarpogrelate in human plasma. Sarpogrelate was extracted from human plasma by solid-phase extraction. Temocapril was used as the internal standard. Heated electron spray ionization mass spectrometry was performed on a TSQ Quantum Ultra MS system. The LC column was a Hypurity C₁₈ and the mobile phase was 2 mM ammonium formate (pH 3.00 ± 0.05):acetonitrile (30:70 v/v). A flow rate of 0.250 mL min^?1 was used. The quantitative analyses were carried out in the positive ion and full scan mode over the mass range m/z 60–500. The capillary, vaporiser temperatures were 325 and 200 °C respectively. The sheath gas pressure, spray voltage, collision energy and tube lense were 40, 3,500 V, 19 V, 198 V, respectively, and the mass spectra of the drugs were recorded by total ion monitoring. Retention times and characteristic mass fragments were recorded and the chosen diagnostic mass fragments were monitored in the mass chromatography mode. Signal intensities of each of the mass fragments: m/z 477 [M + H]⁺ for temocapril, m/z 430 [M + H]⁺ for sarpogrelate, were used for quantification. The calibration curves (the ratio between the peak areas as signal intensities of the drug analyzed and that of the internal standard (temocapril: m/z 477 [M + H]⁺) vs. the concentration of drug) exhibited linearity over the concentration range 5.00–2,500.00 ng mL^?1 human plasma. The recovery and the accuracy were calculated by comparing the peak areas as the signal intensities of each mass fragment for the drug in spiked samples after solid-phase extraction from human plasma to the peak area as the signal intensity of the mass fragment of internal standard sample. The method involves a rapid solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection up to picogram levels with a total run time of 3.0 min only. The method was validated over the range of 5.0–2,500.0 ng mL^?1. The absolute recoveries for sarpogrelate (93.72%) and IS (91.42%) achieved from spiked plasma samples were consistent and reproducible.     

Identification and Determination of Related Substances in Diosmin Bulk Drug and Pharmaceutical Formulations by HPLC and HPLC–MS

Six related substances were detected in diosmin bulk drug substances and products by a newly developed gradient reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection. The chromatographic system consisted of an Intersil Wondasil TM ODS (C 18) column (250 × 4.6 mm; 5 μm). The mobile phase consisted of water/acetic acid 66:6 v/v (solvent A) and methanol (solvent B) using a gradient program at a flow rate of 0.8 mL min^?1 with 345 nm detection and an injection volume of 20 μL. In addition, the linearity, quantitation limit (QL), accuracy, selectivity, robustness and precision were determined. Good linearity was obtained over the concentration range 0.5–200 μg mL^?1 with the coefficient of determination (r ²) of 0.999. The QL was 0.125 μg mL^?1 (relative standard deviation <2.0 %). The major impurities have been resolved and identified using two analytical systems, HPLC and HPLC/electrospray ionization-mass spectrometry operated in a negative ion mode. One of these impurities marked as 7-hexopyranosidal diosmetin was unknown and has not been reported previously. Based on mass spectrometry data the structure of the new impurity was proposed as 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-4H-chromen-7-yl hexopyranoside. The newly developed RP–LC method for quantitative determination of diosmin-related substances was found to be precise, accurate, robust and specific. It has been successfully employed for the quality evaluation of different sources of raw material and generic formulations of diosmin.     

Rapid and Sensitive UPLC-MS–MS for the Determination of Trimetazidine in Human Plasma

A specific and sensitive UPLC-MS–MS was developed for the determination of trimetazidine in human plasma. The sample preparation was based on a single-step liquid–liquid extraction with acetic ether. The chromatographic separation was on a C₁₈ analytical column (50 mm × 2.1 mm, 1.7 μm) with acetonitrile and 10 mM ammonium acetate (30:70, v/v) as the mobile phase, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) applied for detection. The method was linear over the concentration ranges of 0.25–100.00 ng mL⁻¹ for trimetazidine, and the lower limit of quantification (LLOQ) was 0.25 ng mL⁻¹. The intra- and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, this method has been successfully applied to analyze plasma samples from a bioequivalence study with 18 volunteers.

     

Accuracy Profile Theory for the Validation of an LC–MS-MS Method for the Determination of Risperidone and 9-Hydroxyrisperidone in Human Plasma

A sensitive and specific LC–MS-MS method is described for the simultaneous quantification of risperidone and 9-hydroxyrisperidone in human plasma. After extraction with tert-butyl methyl ether, plasma samples were separated on an Atlantis HILIC Silica C18 column (4.6 × 150 mm, 5 μm)with a mobile phase of ammonium formate buffer (10 mM, pH 4.0)/acetonitrile (40/60, v/v). Detection was by MS-MS. The method was fully validated according to the accuracy profile theory. It is based on β-expectation tolerance interval for the total measurement error which includes trueness and intermediate precision. The measurement uncertainty derived from β-expectation tolerance interval was estimated at each of the validation standards. The linearity fitted well over the range of 0.11–26.75 ng mL^?1 for risperidone with an LLOQ of 0.11 ng mL^?1, and for 9-hydroxyrisperidone, at a range of 0.15–37.8 ng mL^?1 with an LLOQ of 0.15 ng mL^?1. The intra- and inter-batch precision of risperidone were <5.71 and 8.22%, respectively. For 9-hydroxyrisperidone, the data were 5.78 and 6.48%. The recoveries were 88.78% (risperidone) and 70.35% (9-hydroxyrisperidone). The developed method was applied to a pharmacokinetic study of risperidone.     

Rapid and Sensitive UPLC-MS–MS for the Determination of Trimetazidine in Human Plasma

A specific and sensitive UPLC-MS–MS was developed for the determination of trimetazidine in human plasma. The sample preparation was based on a single-step liquid–liquid extraction with acetic ether. The chromatographic separation was on a C₁₈ analytical column (50 mm × 2.1 mm, 1.7 μm) with acetonitrile and 10 mM ammonium acetate (30:70, v/v) as the mobile phase, and a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI) applied for detection. The method was linear over the concentration ranges of 0.25–100.00 ng mL^?1 for trimetazidine, and the lower limit of quantification (LLOQ) was 0.25 ng mL^?1. The intra- and inter-day relative standard deviation (RSD) were less than 15% and the relative error (RE) were all within 15%. Finally, this method has been successfully applied to analyze plasma samples from a bioequivalence study with 18 volunteers.     

Liquid Chromatographic Method for Determination of Free and Niosome‐Entrapped Nimodipine in Mouse Plasma and Different Tissues

Abstract

A rapid HPLC method for the quantification of nimodipine in mouse plasma and tissues has been developed in this study, with simple procedure of sample preparation by one‐step protein precipitation. The results of HPLC analysis indicated that linear calibration curves were obtained over the concentration range 0.10–10.00 µg/ml for plasma, 0.10–20.00 µg/g for heart, liver, spleen, kidneys, and brain, and 1.00–200.00 µg/g for lung, respectively. The desirable precision and accuracy were achieved, both intraday and interday for plasma and tissue homogenates. Thus, this newly developed procedure was successfully applicable for determination of nimodipine in mouse plasma and tissues following intravenous administration of free and novel niosome‐entrapped nimodipine.     

High‐Performance Liquid Chromatography Determination of Latanoprost in Pharmaceutical Formulations using UV Detection

Abstract

A simple, fast, and accurate high‐performance liquid chromatography (HPLC) method was developed to determine latanoprost in pharmaceutical formulations. The drug was chromatographed on a C₁₈ column. Eluents were monitored at a wavelength of 210 nm using a mixture of acetonitrile and 0.05 M potassium phosphate buffer pH 3.0 (70:30, (v/v). A linear response (r>0.9998) was observed in the range of 10.0–90.0 µg mL^?1. The method showed good recoveries (average 100.4%) and the relative standard deviations intra‐ and inter‐day were ≤1.0%. The method can be used for quality control assay of latanoprost in raw materials as well as in pharmaceutical formulations.     

Comparative Spectrophotometric,Spectrofluorometric, and High‐performance Liquid Chromatographic Study for the Quantitative Determination of the Binary Mixture Felodipine and Ramipril in Pharmaceutical Formulations

Abstract

Two‐component mixtures of felodipine (FLD) and ramipril (RMP) were assayed by derivative UV spectrophotometry, spectrofluorometry, and high performance liquid chromatography (HPLC). The spectrophotometric methods included a zero‐crossing first‐ and second‐order derivative procedure and a derivative compensation technique for the determination of binary mixtures with overlapping spectra. The spectrofluorometric method was based on first‐ and second‐order derivatives of the emission spectra (zero‐crossing point). Results from these methods were compared with those obtained by an exclusively developed isocratic reversed phase HPLC method. A reversed‐phase Adsorbosil DS analytical column, with methanol‐acetonitrile‐water (50∶30∶20, v/v) mobile phase at a flow rate of 1.5 ml/min, was used with a UV detector. The temperature was set at 25±0.2°C. Results obtained by the spectrophotometric and spectrofluorometric methods were comparable to those obtained by the HPLC method, as far as analysis of variance (ANOVA) test results were concerned. It is concluded that the developed methods are equally accurate, sensitive, and precise; with direct and simple application to pharmaceutical formulations of felodipine and ramipril combination, without interference from common pharmaceutical adjuvants.     

LC–MS Method for the Sensitive Determination of Metoclopramide: Application to Rabbit Plasma, Gel Formulations and Pharmaceuticals

To support real biological sample application, a simple, selective and rapid LC–MS method has been developed and validated for the sensitive determination of metoclopramide in rabbit blood, ex vivo permeation studies and pharmaceutical dosage form. LC–MS analysis was performed isocratically on a Zorbax SB-C₁₈ column with a mobile phase consisting of methanol:ammonium acetate buffer (pH 3.0) 75:25 (v/v) at a flow rate of 0.70 mL min^?1. The outlet of the column was connected to a single quadrupole mass spectrometer with positive mass spectrometric detection. Ions were detected in the positive multiple reaction monitoring mode. The assay was linear over the concentration range of 1.25–200 pg μL^?1 with a limit of detection of 0.077 pg μL^?1 for standard solutions and 2.5–200 pg μL^?1 with a limit of detection of 0.42 pg μL^?1 for serum samples. The method is applicable, covering a variety of pharmaceutical and biological studies. Metoclopramide was extracted from rabbit blood by liquid–liquid extraction using ether as the extraction solvent. The reproducibility of the method was found to be between 0.96 and 1.98 % (RSD) values. The proposed method has been extensively validated for the determination of metoclopramide in all working media. The sample preparations, flow rate and run time of the analytical systems are not time consuming. Moreover, for the stability of metoclopramide, the effect of temperature, UV light, H₂O₂, HCl and NaOH were also investigated.     

A Sensitive LC–ESI–MS–MS Method for the Determination of Huperzine A in Human Plasma: Method and Clinical Applications

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C₁₈ column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL^?1 with a coefficient of correlation (r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL^?1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers.     

Application of Internal Standard for Micro Extraction– Spectrophotometric Determination of Bismuth in Pharmaceutical Formulations

 A micro extraction – spectrophotometric procedure is developed for the determination of bismuth in pharmaceutical formulations. The procedure is based on the extraction of tetraiodobismuthate(III) ion paired with benzyltributylammonium cation into chloroform. The application of Nile Blue as internal standard (IS) enabled good analytical performance for micro-scale analysis. The ratio between the absorbances measured at 491 nm (bismuth complex) and at 632 nm (IS) was taken as the analytical signal. The procedure was carried out in Eppendorf tubes, lowering significantly the use of reagents and the volume of organic solvent. In the calibration range up to 60 mgċl⁻¹, the linear regression coefficient was 0.9999, the CV for 15 mgċl⁻¹ and for 50 mgċl⁻¹ Bi were 1.6% and 0.7% respectively. The results obtained in the analysis of pharmaceutical formulations were in good agreement with the results of EDTA titration method. Received November 25, 1999. Revision February 14, 2000.     

Development and Validation of an RP–LC–UV Method for the Determination of Ondansetron: Application to Pharmaceutical Dosage Forms

A new, simple, rapid, sensitive and specific isocratic RP–LC–UV method was developed and validated for the determination of ondansetron in pharmaceutical dosage forms of orally disintegrating tablets, oral solution and injection. The LC separation was achieved on a Hypersil C4 column (250 × 4.6 mm, 5 μm) using a mobile phase of 50 mM potassium dihydrogen phosphate anhydrous adjusted to pH 3.5 with orthophosphoric acid and acetonitrile (30:70, v/v) at a flow rate of 1.0 mL min^?1 and UV detection at 310 nm. The method was validated for specificity, linearity, precision, accuracy, limit of quantification, limit of detection, robustness and solution stability. The calibration curve was linear over a concentration range of 100–1,000 ng mL^?1 (r ²  = 0.9996) with limit of detection and limit of quantification 50 and 100 ng mL^?1, respectively. The intra-day and inter-day precision and accuracy were between 0.79 and 2.37% and ?0.64 and 1.65%, respectively. The method was successfully applied for analysis of ondansetron in the presence of excipients in commercially available pharmaceutical dosage forms.     

A New Sensitive Reversed‐phase High‐performance Liquid Chromatography Method for the Quantitative Determination of Metoclopramide in Canine Plasma

Abstract

A specific and sensitive high‐performance liquid chromatographic method was developed for the determination of metoclopramide in canine plasma. The procedure involves fast liquid–liquid extraction and analysis on an octadecyl silane (ODS) column. A preliminary pharmacokinetic study was performed by applying the developed method to a single oral administration of metoclopramide (MCP) to a dog. The validation method yielded good results regarding linearity, precision, accuracy, and specificity. The procedure is suitable for separation and quantification of MCP in canine plasma, enough to quantify 0.2 ng/ml when 0.5 ml of plasma is used. This assay procedure might be useful for the pharmacokinetic study of MCP in dogs.     

Development and Validation of TLC—Densitometric Method for the Quantification of a Steroidal Drug,Danazol in its Pharmaceutical Formulations

JPC – Journal of Planar Chromatography – Modern TLC - A reliable and sensitive thin-layer chromatographic (TLC)-densitometric method is developed for the analysis of danazol in its...     

Kinetic Method for the Determination of α‐Methyldopa in Pharmaceutical Preparations: Analytical Procedure and Reaction Mechanism Considerations

Abstract

A reliable and very simple kinetic method is proposed for the determination of α‐methyldopa in pharmaceutical preparations. It is based of the oxidation of α‐methyldopa, a catechol derivative, to quinone, by the ferric ion in the presence of salicylic acid and HCl. The deep blue complex formed between iron(III) and salicylate (λ_max 525 nm) allows the reaction to be watched as absorbance decreases with the reduction of the ferric ions to ferrous with the consequent dissociation of the complex. Four pharmaceutical preparations were analyzed and the results were compared with those obtained with the spectrophotometric United States Pharmacopeia method. The statistical t‐Student and the F‐tests were applied to compare the results obtained with the two independent methods. In all cases complete agreement, in terms of accuracy and precision, was observed with a confidence level of 95% (α=0.05). Considering the four samples analyzed, using the proposed method, and five determinations for each sample, the observed mean relative standard deviation (RSD) is about 0.8%. The concentration range studied was from about 2×10^?4 mol L^?1 to 18×10^?4 mol L^?1 (about 40 µg/mL to 360 µg/mL) in the final solution and it is quite adequate for the analytical procedure at 25.0±0.1°C. A typical coefficient of determination, r², of the calibration curve, is 0.9994. For the kinetic pseudo first order curves a typical observed r² is 0.99998. The Arrhenius activation energy was found to be 84.2±3.4 kJ mol^?1. A schematic mechanism of the reaction is proposed.